Human and other animal cells are presently being frozen, stored, and then thawed as a means of saving these materials for use at a later date. Currently, human embryos are frozen and/or vitrified to accomplish cryopreservation. The terms frozen, vitrification and cryopreservation are interchangeable for the purpose of the subject disclosure.
The problem facing current methods of cryopreservation is the recovering process and then the culturing to implantation for oocyte and embryos, and the resulting actual live birth. Currently, the freezing and thawing of a human oocyte may result in approximately a 10% overall live birth rate, which is well below the actual live birth rate of non-frozen oocyte or embryos. The conditions affecting the oocyte also affect embryos which are frozen and thawed and then implanted. In other words, there is a reduced rate of live birth for the frozen embryos as compared to the non-frozen embryos.
The prior methods for the preparation and cryopreservation of specimens such as embryos, oocyte and stem cells are centered on separate and different solutions and chemical ingredients.
When the time comes to thaw the specimens the current methods utilize a progression of media that contain a combination of solutions used in sequence. Again one may use up to seven different media solutions that are in separate vials. The more common methods are a progression of media reducing the level of sucrose in each and then the last solution will be sucrose free and used for washing. After a specimen is thawed it is then transferred into a generic solution or off the shelf culture media solution used for the normal culturing of oocyte and embryos and do not address any changes in concentrations or the addition of new ingredients to compensate for the freezing and thawing process.
In the current methods of cryopreservation, the specimen is immersed into a series of media and cryoprotectorants whereby the concentration of the cryoprotectorants (CP) is increased until, in some methods, the CP is 50% of the solution. The resulting infusion of the CP into the cells displaces water in the cells and dehydrates the cells during this introduction of the CP. This can be harmful to the cells. The cells are then vitrified and then plunged into liquid nitrogen (LN) at a temperature as low as −180 C. The specimens will then be stored in the LN until they are to be used.
The specimens, when needed, are removed from the LN and then immersed in a series of media used to thaw the specimens. Currently, most of these media contain a solution consisting of a base saline medium, HSA (10-20% of solution) and sucrose as an energy source. In one example Vial #1 will contain 0.85 mM of sucrose. Vials #2 through #4 will decrease the sucrose concentrations stepwise down to 0.10 mM in Vial #4, and then Vial #5 will contain only a base media and HSA, and no sucrose.
The specimens will then be placed into a “generic” culture media until usage. Embryos will be held in this medium until they are transferred into the patient. Currently, embryos used in human IVF will be immersed in several media, embryo culture media, such as P1, HTF, HTF w/SSS and Irvine ECM® (Irvine Scientific), G2.2 through G5 series (Vitrolife), Quinn's Advantage (Sage), Cook® Cleavage and Cook® Blastocyst (Cook IVF), Universal IVF medium®, EmbryoAssist™, BlastAssist™, ISM1 and 2™ and others (MediCult), Sigma Ham's F-10 and MEM 199 (Sigma), and Global® (LifeGlobal). These media have not been modified to accommodate the problems being faced by the specimens in their post thawing development. They are not modified to be different for nonfrozen embryos as differentiated from frozen and then thawed embryos.
The introduction of the CP in the freezing process often results in problems with and damage to the cells, such as dehydration, membrane damage, damage to the spindle, DNA damage, alterations to proteins, stripping of proteins, may alter ingredients, as well as other structural and physical changes and damage. For example, the frozen state can result in damage to the cell walls and to the mitochondria.
The freezing process may introduce other factors such as ice formation during either cooling or warming, and mechanical disruption of cells contributing to the destabilization of membranes. Severe dehydration from the exposure to concentrated CP can cause the structural transitions of biological molecules including lipids and proteins.
It appears that oocytes and embryos may lose or have certain diminished metabolic levels within the oocytes or embryos after the current freezing and thawing procedures, and the current practices do not address these freeze related extractions of certain proteins or nutrients when the CP is extracted or added.
The problems facing the specimens is that they may not recover from the freeze, may not be fully flushed of the cryoprotectorant, may have suffered physical damage to membranes or mitochondria and may not be provided with the correct dosage of the required nutrients or ingredients in order to recover. The problem facing the specimens is that most media used in the freezing and thawing processes are those “generic” media which are used for the culture and development of nonfrozen specimens, and which have different requirements.
In the current methods used for cryopreservation techniques, the specimens are not “cultured” or “aided” in the process when they are going to be placed into cryopreservation or thawed out of cryopreservation. The users utilize “generic” media, as they do in post thawing, as mentioned earlier, and do not add or alter any ingredients which may better prepare the specimen for cryopreservation, thawing or recovery.
The specimens are placed into vitrification kits, which do not address pre-vitrification or post vitrification conditions. Currently, embryos used in human IVF will be immersed in several vitrification solutions as part of a sequential kit, such as Embryo and Blastocyst Freeze and Thaw Kit, Blastocyst Vitrification Freeze and Thaw and others (Irvine Scientific), G-FreezeKit Blast™, RapidVit™ Cleave, RapidWarm™ Cleave and others (Vitrolife), Quinn's Advantage® embryo freeze kit, Quinn's Advantage® blastocyst freeze kit (Sage), Blastocyst Cryopreservation™ and Blastocyst Vitrification™ (Cook IVF), OocyteFreeze™ and Thaw™, BlastFreeze™ and BlastThaw™ and others (MediCult). These vitrification solutions, used in sequence do not contain a solution or method of immersing the embryos in a specially prepared, modified media to accommodate the problems being faced by the specimens of their pre-vitrification or post thawing procedures. They do not modify the media, solutions or methods so as to be different for nonfrozen embryos as differentiated from frozen, and then thawed, embryos.